Method of killing microorganisms



Patented Mar. 23, 1926.

ururso s'rArns' tenses PATENT OFFICE.

WILHELM RUPPEL, OF BERLIN, GERMANY, ASSIGNOR, BY MESNE ASSIGNMENTS, TO

AMERICAN ELECTRO-OSMOSIS CORPORATION, OF NEW YORK, N. Y., A CORPORA- TION OF DELAWARE.

METHOD OF KILLING MICROORGANISMS.

No Drawing.

T all whom it may concern- Be it known that I, WILHELM RUPPEL, doctor med., professor, residing at Tiergartenstrasse 13-, Berlin, Germany, have invented certain new and useful Improvements in Methods of Killing Microorganisms, for which applications have been filed in Germany, Aug. 19, 1919; Austria, Dec. 12, 1919; Poland, June 9, 1920; Netherlands, July 22, 1920; Sweden, July 8, 1920; Czechoslovakia, July 3, 1920; Belgium, Aug. 3, 1920; Switzerland, July, 23, 1920, and of which the following is a specification.

The application of electric current for killing micro-organisms, or depriving them of their pathogenesy, that is for sterilizing,

has had but slight success and has remained without practical importance. It was gener- 0 ally supposed that electric current could not be used for sterilizing and that any result attained was due. not to the electric current as such, but to the action on the organisms of products set free by electrolysis. I have found however that the electric current as such. has a powerful effect in killing microorganisms of any kind or depriving them of their pathogenesy, provided that the conditions under which the current acts on the micro-organisms are correctly selected.

I have found that a continuous current of certain strength will kill micro-organisms, while voltages merely have only a slight or no effect on them. The current strength necessary for killing micro-organisms varies with the kind of organisn'is and its capacity for resistance. The destructive process is generally a function of the current strength and of the duration of action of the current as well as of the specific resistance capacity of the kind of micro-organisms. Microorganisms having very small resistance require only small current strength, or at a certain current strength are quickly killed, while to obtain the same effect on bacteria having high resistance and particularly with bacteria having What is called a permanent form the same current strength has a much slower action. In most cases a current of 10-12 amperes at -30 volts, over an effective surface of about 400 sq. ems. will kill within two hours all c -0 's 11 m hi h I ha e investiga ed.

aid

Application filed August l, 1921 Serial No. 489,837.

In order to exclude any subsidiary action of electrolytic products and also the action of high temperature from the start, the sterilization is preferably carried out between suitable diaphragrns, that is, best of all, in a socalled three-cell apparatus.

Such three-cell apparatus may consist of a box of suitable material, partitioned longitudinally into three chambers by suitable semi-permeable diaphragms in a manner well understood by those skilled in the art. The material to be sterilized is placed in the middle chamber. The diaphragms carry on that face which is not within the middle chamber, electrodes of net-work or lattice-work made of suitable material, by means of which electric current can be supplied at the diaphragms to the middle chamber. The outer chambers, that is the cathode and anode'chambers are filled with water, which may be kept in circulation by suitable devices.

The material to be sterilized may be either in solid or in liquid form. If solids, or solids saturated with liquid, are to be subjected to the sterilizing action of an electric current, they may either be saturated with a solution of common salt or other electrolyte and placed in the middle chamber of the aforesaid apparatus, or placed in the middle chamber and the remaining space around them filled with salt solution or another electrolyte of suitable strength. This strength must be high enough for attainment of the necessary current strength at the voltage employed.

In order to follow quite exactly the action of the electric current on micro-organisms, I performed experiments with pure cultures of bacteria which. were cultivated, namely 1. streptococci, staphylococci, meningo cocci, pneumococci, and common, non-pathogenie atmospheric cocci;

2. Diphtheria bacilli, dysentery bacilli, tubercular bacilli. typhus bacilli, cholerabzlicilli, the Bacillus 00h communal? and the li (e;

3. Spore-bacteria, such as splenic gan- 4:. Several germs belonging to the class of so-called ultra-visible or iilterable kinds of virus, as the eXciter of the cow-pox and lyssa.

Itwas ascertained that the several kinds of cocci and bacilli named under (1) and (2) differ considerably in their power to resist the electric current. Streptococci, staphylococci and pneumococci were killed in 10-20 minutes under the action of a current of 10-12 amperes passing through a cross sectional area of about 400 square centimeters, when in suspension (not too concentrated) in common salt solution free from proteid. .The same was also found to be true, with diphtheria bacilli, bacilli of swine fever and with the group of the bacilli of septiccemia. Dysentery bacilli have more resistance, for, with the same strength of current, they required three-quarters of an hour, and the bacilli from this group which. have the greatest resistance are the tuber; cular bacilli which lost their power of development only after an hours treatment with the current in question. The sporobacilli of group required one and a half to two hours for complete destruction. The

irus class, (l), showed a similar difference in resistance, the virus of cow-pox having much more resistance than that of the virus of lyssa.

These facts about pure cultures of the various micro-organisms having been ascertained, I made experiments to sterilize material containing bacteria by means of electric current. Decayed meat in pieces of 6 sq. cm. surface, or 1 sq. cm. thick,

together with a common salt solution of 1 per cent strength was put into the middle chamber of the apparatus, and treated in the same manner with a current of 12 amperes for two hours, and it was found that the meat was rendered completely odouriess and all putrefactive bacteria were killed.

Since in the apparatus described the electrclyte is very quickly impoverished by the electric current, in order to maintain the current strength, additional electrolyte must be added from time to time to the middle compartment. The apparatus has further the advantage, that when the sterilization is finished the surplus electrolyte can be removed electro-osniot-ically. By flow of water through the anode and cathode compartments the temperature can be regulated. On an average a temperature of 18-25 C. is preferably maintained. In sterilizing liquids it is advantageous to keep the liquid in the middle compartment in constant motion by stirring, or better, byintroducing indifferent gases such as nitrogen or arhon dioxide to agitate the liquid.

As an example of the treatment of liquids there may becited the sterilization of a diphtheria serum preparation which wasiififected with numerous germs of various kinds. By adding common salt solution the 'serum is first brought to a salt concentration of 1.2 per cent. Then it was put into the middle chamber of the apparatus and treated for two hours with a constant current of amperes strength, at volts pressure, the temperature being kept at 20 C. After this time the serum, which before the operation contained 10,000 germs per cc., was found to be completely sterilized. without any apparent alteration and without diminution of its antitoxin content.

The process is especially suitable for sterilizing unstable material easily altered by heat and chemical influences since the temperature may be kept down and the products formed by electrolysis are kept from the material being sterilized.

Furthermore the invention can be applied for the manufacture of specific vaccines. I have found that by subjecting pure cultures of micro-organisms to this method, suspensions of dead bacteria are obtained which are exceptionally suitable for the manufacture of specific vaccines.

The manufacture of specific vaccine from bacteria has hitherto consisted in separating, by centrifugal force, thebacteria from pure cultures of bacteria, washing these bacteria with isotonic common salt solution, then suspending them in isotonic salt solution and killing the bacteria by action of high te1nperature (56-60 C.) or by action of disinfectants (phenoh tricresol, aether). From the emulsion of killed bacteria, dilutions corresponding with a certain number of germs in 1 cc. are made and used directly as vaccine. This method has the considerable disadvantage, that heating to high temperature or even action of disinfectants damages considerably the immunizing capacity of the kinds of bacteria in question. (in this account the results obtained by using the vaccines, for instance, the typhus and cholera vaccine so formed, have been very poor.

This fact has already been recognized and attention has frequently been called to the damaging influence of temperature and chemicals. In consequence many attempts have been made to kill the bacteria in question in a'inore elfective manner. For a temperature of 60 C, which was previously used for the purpose, a temperature of 56 C. has been recently substituted, and among chemical agents all strong disinfectants such as cresol and phenol have been rejected, eether alone being regarded as a suitable effective agent.

I have found that the bacteria killed by means of an electric current according to my invention have an immunizing capacity greatly exceed ng that of the bacteria killed bf l eatfor-cl enncals. I found for example that 1 cc. of a vaccine which was made from swine fever bacilli killed by heating to 56C. delayed for only few days the death of white mice which, eleven. days after being inoculated, were infected with the virulent fever bacilli, while 0.01 cc. of a vaccine made from the fever bacilli killed by an electric current according to my invention, prevented the death of white mice infected with the virulent culture introduced into them eleven days after inoculation. Ofcourse, both vaccines were made from the same number of'germs by enact counting of the germs. Like good results were obtained with streptococci. pneumococci, typhus bacilli, diphtheria bacilli and many others.

I have further discovered that for each kind of bacteria there is a certain limiting current strength, below which the bacteria are not killed even if the action of the current on themicro-organisms is continued for a very long time. I have also found that electric currents, the strength of which is not sufiicient to kill bacteria, will still bring about certain modifications in the microorganisms whereby the bacteria in question lose their pathogenity for experimental ani-' mals, although on artificial nutrients they remain fully capable of development. A like effect can be obtained by allowing current of astrength which is sufficient for killing the micro-organisms to act on them for an insufficient time. i

For example, I took pneumococci culture of strong pathogenity for white mice and guinea-pigs and cultivated it on a solid nutrient. The cocci, after they had attained a strong growth, where separated from the nutrient by suspension in physio logical common salt solution and made'into a fine emulsion by long shaking with the common salt solution. This emulsion was first of all subjected for one and a half hours in the centre chamber of a three-cell apparatus to the action of an electric current of 1012 amperes, the effective electrode surface being 400 sq. ems. San'iples taken showed that the pneumococciwere' completely killed in this time when using such current strength. Then an emulsion of live pneumococci was prepared in the same manner and subjected to a current of the same strength, but for only half an hour. The cocci were then transferred to suitable nutrient and were found to be fully capable of development and after 24 hours showed strong growth, but as the inoculation therewith of white mice and guinea-pigs showed, they had lost all pathogenity for this class of animals. Before the electro-osmotic treatment, 0.01 cc. of the emulsionof the pneumococci was sufficient to kill white mice and 0.1 cc. to killguinea-pigs of 950 grins. weight within three or four days with ce ainty. After half an hours action of the .to say, it had lost its paraog'enity for experimental anin'ials, from which it must be concluded that the electric current has permanently affected the culture. Up to the present, at least, I have not succeeded in restoring to a-culturo so affected by the electro-osmotic treati'i'ient its original animal pathogenity by cultivating it on artificial nutrients.

Tubercle bacilli of humans or of bovinus kind behave exactly similarly. A strong growth of tubercle bacilli was separated from its nutrient, emulsified in common salt solution and subjected to the action of an electric current in the central chamber of the threecell apparatus. The culture subjected to the action of a current of 10 amperes, was so far killed after two hours, that propagation on an artificial nutrient was no longer possible. I found, however, that if the electric current acts on the culture emulsion for only one to one and a half hours, then on a suitable nutrient a strong growth of tubercular bacilli was obtained. l hen this culture was used for inoculating guineapigs (sub-cutaneous or intraperitoneal) it proved non-pathogenic, that is, it no longer caused tubercular sickness in the animals. Guinea-pigs which were subcutaneously or intraperitoneally infected with considerable doses of the. culture so altered by the electric current showed no externally visible symptoms of tubercular sickness even after 68 weeks. At the place of injection no external alteration could be seen, and no glandular swellings ensued. On killing and dissecting the animals it was proved that, beyond immaterial swelling at the place of injection, absolutely no pathologic change had occur red, that is, the internal organs were per fectly normal.

In many cases it will be of advanta to use, for making these vaccines, instead or the killed culture material, micro-organisms thus mitigated by clectro-osn otic treatment. The technique of making these vaccines is the same as when the cultures with killed micro-organisms are used.

ll hile I have described my in'i irovements in great detail and with respect to certain examples, I do not desire to be limited to such details or examples since many changes may be made and my improvements may be embodied in widely different forms without departing from the spirit and scope thereof in their broader aspects. Hence-I desire to llll) lit) liHl

cover all modifications and forms coming within the language or scope of any one or more of the appended claims.

lVhat I claim as new and desire to secure by Letters Patent, is;

1. A method of killing micro-organisms with aid of an electric current, which conin subjecting the micro-organisms to the action of the electric current between diaphragms which separate the electrode. from the micro-organisms.

2. A method of killing microorganisms with aid of an electric current, which consists in subjecting the micro-organisms to the action of the electric current between diaphragms which separate the electrodes from the micro-organisms,the electric current being 01 high strength and low tension.

3. A method of killing micro-organisms by subjecting the micro-organisms to the action of an electric current between dia phragms in a three chamber cell, the electrode chambers being cooled during the electro-osmosis.

A method for killing micro-organisms by subjecting the micro-organisms to the action oi an electric current between diaphragms, the current strength being kept substantially constant by the addition of salt to the medium surrounding the microorganisms.

A method of treating microorganis1ns consisting in that the micro-organisms are subjected to the action of a continuous electric current between diaphragms with the micro-organisms between the diaphragms.

A process for making vaccines from cultures of miere-organisms, which consists in treatingthe micro-or anisms, with aid of continuous electric current flowing between diaphragms with the micro-organisms between the diaphragms and making the so-treated micro-organisms into emulsions with common salt solution.

7. The process for making specific vaccines from pure cultures of micro-organisms which consists in subjecting the pure cultures micro-organisms to the action of an electric current flowing between diaphragms, with the micro-organisms between the diaphragms, theelcctro-osmosis being continued until the culture is deprived of its pathogenesy, and then making the cultures into emulsions with common salt solution.

A process for making Vaccines from cultures of micro-organisms which consists in subjecting the micro-organisms to the action of an electric current in an electric cell wherein the culture is separated from the electrodes by diaphragms until the culture is deprired of its pathogenesy.

9. A process for making Vaccines from cultures of micro-organisms which consists in killing the 'micro-organisms' by aid or" electric current flowing between diaphragms in an electric cell with the micro-organisms between the diaphragms and making the killed micro-organisms into emulsions with common salt solution.

10. The method of treating micr0-organisms which consists in subjecting them to the action of a direct electric current in an electric cell wherein the micro-organisms are separated from the electrodes of the cell by diaphragms.

11. The method of treating micro-organisms which consists in subjecting them to the action of an electric current in an electric cell wherein the micro-organisms are separated from the electrodes by diaphragms and the micro-organisms are surrounded by a liquid electrolyte, the current being continued until the organisms are deprived of their pathogenesy.

li The method ot treating micro-orgair isms which consists in subjecting them to the action of an electric current in an elec tric cell wherein the micro-organisms are separated from the electrodes by diaphragms and the micro-organisms are surrounded by a liquid electrolyte, the current being continued until the organisms are de prived of their pathogenesy and fresh electrolyte being added during the electroosmotic treatment to maintain the current strength.

13. The method of treating micro-organisms which consists in subjecting them to the action of an electric current in an electric cell wherein the micro-organisms are separated from the electrodes by diaphragms and the micro-organisms are surrounded by a liquid electrolyte, the current being continued until the organisms are deprived of their pathogenesy, and the temperature of the cell being maintained below the point where the micro-organisms would be killed by heat.

1 The method of treating micro-organisms which consists in subjecting them to the action of an electric current in an electric cell wherein the micro-organisms are separated from the electrodes by diaphragms, and the micro-organisms are surrounded by a liquid electrolyte, the current being continued until the organisms are deprived of their pat-ho enesy, and the strength of the current being about to 12 amperes or more per 400 square centimeters of electrode surface.

15. The method of treating micro-organisms which consists in subjecting them to the action of an electric current in an electric cell wherein the micro-organisms are separated from the electrodes by diaphragms and the micro-organisms are surrounded by a liquid electrolyte, the current being continued until the organisms are deprived of their pathogenesy, fresh electrolyte being added during the electroosmotic treatment to maintain the current strength, and the temperature of the cell being maintained below the point where the micro-organisms would be killed by heat.

16. A pro ess for making vaccines from cultures of micro-organisms which consists in subjecting the micro-organisms to the action of an electric current in an electric cell wherein the culture is separated from the electrodes by diaphragms until the culture is deprived of its pathogencsy, the temperature of the cell being maintained below the point where the micro-organisms would he killed by heat.

17. A process for making vaccines from cultures of micro-organisms which consists in subjecting the micro-organisms to the action of an electric current in an electric cell wherein the culture is separated from the electrodes by diaphragms until the culture is deprived of its pathogenesy, the temperature of the cell being maintained below the point where the micro-organisms would be killed by heat, and fresh electrolyte being added during the electroosmotic treatment to maintain the current strength.

18. A process for making vaccines from cultures of micro-organisms which consists in subjecting the micro-organisms to the action of an electric current in an electric cell wherein the culture is separated from the electrodes by diaphragms until the culture is deprived of its pathogenesy, fresh electrolyte being added during the electroosmotic treatment to maintain the current strength, the temperature of the cell being maintained below the point where the micro-organisms would be killed by heat, and the strength of the current being about ture is deprived of its pathogenesy and the strength of the current being about 10 to 12 ainperes or more per lOO square cent1- meters of electrode surface.

20. The method of treating 1nicro-organ-,

isms which consists in subjecting the microorganisms to the action of an electric current in an electrolytic cell, the strength of the current being about 10 to 12 amperes or more per 400 square centimeters of electrode surface.

21. The method of treating micro-organ isms which consists in subjecting the microorganisms to the action of an electric current in an electrolytic cell fresh electrolyte being added during the electro-osmotic treatment to maintain the current strength and the temperature of the cell being maintained below the point where the microorganisms would be killed by heat.

22. The method of rendering microorganisms non-pathogenic which consists in passing an electric current through a medium containing the microorganisms the current being so great in amperage and low in voltage that the micro-organisms are rendered non-pathogenic by the passage of the current instead of by heat generated therein or by the formation of germicidal electrolytic products produced.

In testimony whereof I affix my signature.

Pnor. DR. lVILI-IELM RUPPEL.

ill

Certificate of Correction.

It is hereby certified that in Letters Patent No. 1,577,659, granted March 23, 1926, upon the application of Wilhelm Ruppel, of Berlin, Germany, for an improvement in Methods of Killing Microorganisms, errors appear in the printedv specification requiring correction as follows: Page 1, line 33-, after the Word While insert the Word high; page 3, line 38, for the Word Where read were,

and line 84, for the Word humans read hmnames; and that the said Letters Patent should be read With these corrections therein that the same may conform to the record of the case in the Patent Oflice.

Signed and sealed this 27th day of April, A. D. 1926.

[em] M. J. MOORE,

Acting Gammz'ssz'oner of Patents. 

